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Budna, Joanna, Artur Bryja, Piotr Celichowski, Rotem Kahan, Wiesława Kranc, Sylwia Ciesiółka, Marta Rybska et al. "Genes of cellular components of morphogenesis in porcine oocytes before and after IVM". Reproduction 154, n.º 4 (octubre de 2017): 535–45. http://dx.doi.org/10.1530/rep-17-0367.
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Proper oocyte maturation in mammals produces an oocyte capable of monospermic fertilization and embryo preimplantation. The cumulus-oocyte complexes (COCs), surrounding an oocyte, play a significant role in oocyte maturation. During this process, when the COCs undergo cumulus expansion wherein tightly compact cumulus cells (CCs) form a dispersed structure, permanent biochemical and molecular modifications occur in the maturing oocytes, indicating that the gene expression between immature and mature oocytes differs significantly. This study focuses on the genes responsible for the cellular components of morphogenesis within the developing oocyte. Brilliant cresyl blue (BCB) was used to determine the developmental capability of porcine oocytes. The immature oocytes (GV stage) were compared with matured oocytes (MII stage), using microarray and qRT-PCR analysis to track changes in the genetic expression profile of transcriptome genes. The data showed substantial upregulation of genes influencing oocyte’s morphology, cellular migration and adhesion, intracellular communication, as well as plasticity of nervous system. Conversely, downregulation involved genes related to microtubule reorganization, regulation of adhesion, proliferation, migration and cell differentiation processes in oocytes. This suggests that most genes recruited in morphogenesis in porcine oocytein vitro,may have cellular maturational capability, since they have a higher level of expression before the oocyte’s matured form. It shows the process of oocyte maturation and developmental capacity is orchestrated by significant cellular modifications during morphogenesis.
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Pedersen, Hanne Skovsgaard, Peter Løvendahl, Knud Larsen, Lone Bruhn Madsen y Henrik Callesen. "Porcine oocyte mtDNA copy number is high or low depending on the donor". Zygote 24, n.º 4 (18 de diciembre de 2015): 617–23. http://dx.doi.org/10.1017/s0967199415000611.
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SummaryOocyte capacity is relevant in understanding decreasing female fertility and in the use of assisted reproductive technologies in human and farm animals. Mitochondria are important to the development of a functionally good oocyte and the oocyte mtDNA copy number has been introduced as a useful parameter for prediction of oocyte competence. The aim of this study was to investigate: (i) if the oocyte donor has an influence on its oocyte's mtDNA copy number; and (ii) the relation between oocyte size and mtDNA copy number using pre- and postpubertal pig oocytes. Cumulus–oocyte complexes were collected from individual donor pigs. The oocytes were allocated into different size-groups, snap-frozen and single-oocyte mtDNA copy number was estimated by quantitative real-time PCR using the genes ND1 and COX1. Results showed that mean mtDNA copy number in oocytes from any individual donor could be categorized as either ‘high’ (≥100,000) or ‘low’ (<100,000) with no difference in threshold between pre- and postpubertal oocytes. No linear correlation was detected between oocyte size and mtDNA copy number within pre- and postpubertal oocytes. This study demonstrates the importance of the oocyte donor in relation to oocyte mtDNA copy number, irrespectively of the donor's puberty status and the oocyte's growth stage. Observations from this study facilitate both further investigations of the importance of mtDNA copy number and the unravelling of relations between different mitochondrial parameters and oocyte competence.
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Walker, Bailey N. y Fernando H. Biase. "The blueprint of RNA storages relative to oocyte developmental competence in cattle (Bos taurus)". Biology of Reproduction 102, n.º 4 (26 de enero de 2020): 784–94. http://dx.doi.org/10.1093/biolre/ioaa015.
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Abstract From the time oocytes leave quiescence, there are constant microenvironmental influences contributing to development, thus acquiring developmental competence is not a simple, linear phenomenon. During folliculogenesis, oocytes experience many morphological and cytological changes that contribute toward the acquisition of developmental competence, a process defined by an oocyte’s ability to progress through folliculogenesis, be fertilized, undergo cleavage, and develop into an embryo. Many factors, such as ovarian follicle size, cow age, and the morphology of the cumulus–oocyte complex, have been extensively investigated to understand this process. In parallel to aiding in the understanding of oocyte biology, these features have been used to characterize an oocyte’s ability to achieve competence. In addition, oocytes undergo intense gene transcription and protein translation to accumulate the maternal stores. When the oocyte is fully grown, most genes are transcriptionally inactive, and the chromatin is densely compacted. More recently, RNA profiling has been used to further define the transcriptional parameters that are associated with oocyte development. Here, focusing on cattle, we provide an overview of the experimental models commonly used to understand the underlying biology related to oocyte developmental competence. We compiled public data and showed that cattle oocytes can express over 15 000 protein-coding genes, suggesting a complex transcriptome landscape. Surprisingly, less than 2% of the expressed genes have been linked to developmental competence. The identification of the gene products that contribute to oocyte development, and understanding their biological function, are a vital component of our quest toward defining oocyte developmental competence at the molecular level.
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Ritter, Lesley J., Satoshi Sugimura y Robert B. Gilchrist. "Oocyte Induction of EGF Responsiveness in Somatic Cells Is Associated With the Acquisition of Porcine Oocyte Developmental Competence". Endocrinology 156, n.º 6 (1 de junio de 2015): 2299–312. http://dx.doi.org/10.1210/en.2014-1884.
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Abstract Oocytes progressively acquire the competence to support embryo development as oogenesis proceeds with ovarian folliculogenesis. The objectives of this study were to investigate oocyte-secreted factor (OSF) participation in the development of somatic cell epidermal growth factor (EGF) responsiveness associated with oocyte developmental competence. A well-established porcine model was employed using oocytes from small (<4 mm) vs medium sized (>4 mm) antral follicles, representing low vs moderate developmental competence, respectively. Cumulus-oocyte complexes (COCs) were treated in vitro with inducers of oocyte maturation, and cumulus cell functions and oocyte developmental competence were assessed. COCs from small follicles responded to FSH but, unlike COCs from larger follicles, were incapable of responding to EGF family growth factors known to mediate oocyte maturation in vivo, exhibiting perturbed cumulus expansion and expression of associated transcripts (HAS2 and TNFAIP6). Low and moderate competence COCs expressed equivalent levels of EGF receptor (EGFR) mRNA; however, the former had less total EGFR protein leading to failed activation of phospho-EGFR and phospho-ERK1/2, despite equivalent total ERK1/2 protein levels. Native OSFs from moderate, but not from low, competence oocytes established EGF responsiveness in low competence COCs. Four candidate recombinant OSFs failed to mimic the actions of native OSFs in regulating cumulus expansion. Treatment with OSFs and EGF enhanced oocyte competence but only of the low competence COCs. These data suggest that developmental acquisition by the oocyte of capacity to regulate EGF responsiveness in the oocyte's somatic cells is a major milestone in the oocyte's developmental program and contributes to coordinated oocyte and somatic cell development.
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Virant-Klun, Irma, Katja Knez, Tomaz Tomazevic y Thomas Skutella. "Gene Expression Profiling of Human Oocytes Developed and MaturedIn VivoorIn Vitro". BioMed Research International 2013 (2013): 1–20. http://dx.doi.org/10.1155/2013/879489.
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The quality of the human oocyte determines the success of fertilization and affects the consequent embryo development, pregnancy and birth; it therefore serves as a basis for human reproduction and fertility. The possibility to evaluate oocyte quality in thein vitrofertilization programme is very limited. The only criterion which is commonly used to evaluate oocyte quality is its morphology. There is a mass of oocytes in thein vitrofertilization programme which are not fertilized in spite of normal morphology. In the past, several attempts focused on oocyte gene expression profiling by different approaches. The results elucidated groups of genes related to the human oocyte. It was confirmed that some factors, such as oocytein vitromaturation, are detectable at the molecular level of human oocytes and their polar bodies in terms of gene expression profile. Furthermore, the first genetic evaluations of oocyte-like cells developedin vitrofrom human stem cells of different origin were performed showing that these cells express some genes related to oocytes. All these findings provide some new knowledge and clearer insights into oocyte quality and oogenesis that might be introduced into clinical practice in the future.
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Němeček, David, Markéta Dvořáková, Ivona Heroutová, Eva Chmelíková y Markéta Sedmíková. "Anti-apoptotic properties of carbon monoxide in porcine oocyte duringin vitroaging". PeerJ 5 (6 de octubre de 2017): e3876. http://dx.doi.org/10.7717/peerj.3876.
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If fertilization of matured oocyte does not occur, unfertilized oocyte undergoes aging, resulting in a time-dependent reduction of the oocyte’s quality. The aging of porcine oocytes can lead to apoptosis. Carbon monoxide (CO), a signal molecule produced by the heme oxygenase (HO), possesses cytoprotective and anti-apoptotic effects that have been described in somatic cells. However, the effects of CO in oocytes have yet to be investigated. By immunocytochemistry method we detected that both isoforms of heme oxygenase (HO-1 and HO-2) are present in the porcine oocytes. Based on the morphological signs of oocyte aging, it was found that the inhibition of both HO isoforms by Zn-protoporphyrin IX (Zn-PP IX) leads to an increase in the number of apoptotic oocytes and decrease in the number of intact oocytes during aging. Contrarily, the presence of CO donors (CORM-2 or CORM-A1) significantly decrease the number of apoptotic oocytes while increasing the number of intact oocytes. We also determined that CO donors significantly decrease the caspase-3 (CAS-3) activity. Our results suggest that HO/CO contributes to the sustaining viability through regulation of apoptosis duringin vitroaging of porcine oocytes.
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Liu, Rui-Hua, Yong-Hai Li, Li-Hong Jiao, Xiao-Ning Wang, Hong Wang y Wei-Hua Wang. "Extracellular and intracellular factors affecting nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles". Zygote 10, n.º 3 (agosto de 2002): 253–60. http://dx.doi.org/10.1017/s0967199402002332.
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Nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles were examined. Oocyte-cumulus complexes were collected from small (1-2 mm in diameter), medium (3-6 in diameter) and large (7-8 mm in diameter) follicles and cultured in a modified tissue culture medium 199 for 44 h. Nuclear maturation was evaluated after orcein staining, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay. Oocyte diameter, cumulus morphology, steroid hormones and glutathione in the follicular fluid (FF), were also examined. Significantly higher proportions of oocytes collected from large and medium follicles reached metaphase II than did oocytes from small follicles. Oocytes from small follicles also had a smaller size. GSH content was significantly higher (p < 0.05) in oocytes from large (14.24 ± 2.1 pmol/oocyte) and medium (13.69 ± 1.5 pmol/oocyte) follicles than in oocytes from small (9.44 ± 1.28 pmol/oocyte) follicles just after collection. After maturation, oocytes from medium follicles had a higher GSH concentration than oocytes from small follicles. It was found that between 49.7 ± 5.18 nM and 52.25 ± 0.78 nM GSH was present in FF but there was no statistical difference between follicle sizes. A significantly higher (p < 0.001) estradiol level was present in FF from large follicles (299.2 ± 68.6 ng/ml) than from medium (40.0 ± 6.4 ng/ml) and small (41.2 ± 3.7 ng/ml) follicles. Progesterone concentrations in FF from large (281.6 ± 45.9 ng/ml) and medium (267.5 ± 38.6 ng/ml) follicles were significantly higher than that (174.7 ± 22.0 ng/ml) from small follicles. These results indicate that the oocyte's ability to accumulate intracellular GSH during maturation, and extracellular steroid hormones and cumulus cells, affect the competence of porcine oocytes to undergo nuclear and cytoplasmic maturation.
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Zuccotti, Maurizio, Anna Piccinelli, Nicola Marziliano, Silvia Mascheretti y Carlo Alberto Redi. "Development and loss of the ability of mouse oolemma to fuse with spermatozoa". Zygote 2, n.º 4 (noviembre de 1994): 333–39. http://dx.doi.org/10.1017/s096719940000215x.
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SummaryTo further our Knowledge on the mechanisms and molecules involved in mouse sperm–oocyte plasma membrane interaciton, exteraction, experiments were carried out to determine the stage during oogenesis at which an oocte acquires the capacity ot fuse with acrosome-reacted sperm. Zona-ferr oocytes 10 μm in diametes do not fuse with sperm. Oolemma fusibility is first acquired when the oocyte reaches about 20μm in diameter. Fusibility is maniatained even after fertilisation has accurred and is lost completely by the 4–cell stage.
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Talreja, Deepa, Chirag Gupta, Hrishikesh Pai y Nandita Palshetkar. "Oocyte Vitrification: A Comparative Analysis Between Fresh and Cryopreserved Oocytes in an Oocyte Donation Program". Fertility & Reproduction 02, n.º 01 (marzo de 2020): 9–13. http://dx.doi.org/10.1142/s2661318220500024.
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Background: Oocyte Cryopreservation has become an important part of infertility treatment for various reasons such as fertility preservation in women going for oncological treatment; in oocyte donation cycles; in eliminating several religious, ethical, and legal concerns of embryo freezing and in women who wish to delay childbirth. The newer ”vitrification” technique for freezing has further improved the success rates for actual conception than the earlier method of slow freezing. A successful oocyte freezing program can help in establishment of oocyte banks, which would help to provide compatible oocytes immediately, thus would eliminate the several problems of fresh donor cycles. Methods: In this retrospective observational study, total 60 oocyte donation cycles were included (38 were fresh and 22 were vitrified oocytes cycle, respectively). After a thorough screening, controlled ovarian hyperstimulation for donors was performed using flexible antagonist protocol. All mature oocytes were allocated into “vitrified oocytes” and “fresh oocytes” groups. Vitrification technique using Cryotop method was used for oocyte freezing. Both clinical and laboratory outcomes of vitrified and fresh oocytes in donor cycles were compared. Results: A total of 600 oocytes (226 “vitrified oocytes” and 374 fresh oocytes), were studied. After warming 218 oocytes survived resulting in survival rate of 96.4%. Fertilization rate and embryo formation rate was 86.2% and 93.6%, respectively. Results of frozen-thawed oocyte donor cycles were compared with fresh donation cycles. For fresh oocyte group, fertilization rate and embryo formation rate was 83.4% and 92.6%, respectively. On comparing clinical outcomes, clinical pregnancy rate was 60.5% in fresh group and 63.6% in vitrified group. Conclusions: Both clinical and laboratory results obtained in the study suggest that oocyte cryopreservation can be performed with reproducible success, thus vitrification technique can be provided as a useful tool for achieving highly successful outcomes in an oocyte donor program.
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Tharasanit, T., S. Colleoni, G. Lazzari, B. Colenbrander, C. Galli y T. A. E. Stout. "Effect of cumulus morphology and maturation stage on the cryopreservability of equine oocytes". Reproduction 132, n.º 5 (noviembre de 2006): 759–69. http://dx.doi.org/10.1530/rep.1.01156.
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Oocyte cryopreservation is a potentially valuable way of preserving the female germ line. However, the developmental competence of cryopreserved oocytes is presently poor. This study investigated whether the morphology of the cumulus complex surrounding an immature equine oocyte and/or the oocyte’s stage of maturation affect its cryopreservability. Compact (Cp) and expanded (Ex) cumulus oocyte complexes (COCs) were vitrified either shortly after recovery (germinal vesicle stage, GV) or after maturation in vitro (IVM); cryoprotectant-treated and -untreated non-frozen oocytes served as controls. In Experiment I, oocytes matured in vitro and then vitrified, or vice versa, were examined for maturation stage and meiotic spindle quality. Cp and Ex COCs vitrified at the GV stage matured at similar rates during subsequent IVM (41 vs 46% MII), but meiotic spindle quality was better for Cp than Ex (63 vs 33% normal spindles). Vitrifying oocytes after IVM resulted in disappointing post-warming spindle quality (32 vs 28% normal for Cp vs Ex). In Experiment II, oocytes from Cp and Ex COCs vitrified at the GV or MII stages were fertilized by intracytoplasmic sperm injection (ICSI) and monitored for cleavage and blastocyst formation. Oocytes vitrified prior to IVM yielded higher cleavage rates (34 and 27% for Cp and Ex COCs) than those vitrified after IVM (16 and 4%). However, only one blastocyst was produced from a sperm-injected vitrified–warmed oocyte (0.4 vs 9.3% and 13% blastocysts for cryoprotectant-exposed and -untreated controls). It is concluded that, when vitrification is the chosen method of cryopreservation, Cp equine COCs at the GV stage offer the best chance of an MII oocyte with a normal spindle and the potential for fertilization; however, developmental competence is still reduced dramatically.
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Curnow, E. C., J. P. Ryan, D. M. Saunders y E. S. Hayes. "Developmental potential of bovine oocytes following IVM in the presence of glutathione ethyl ester". Reproduction, Fertility and Development 22, n.º 4 (2010): 597. http://dx.doi.org/10.1071/rd09228.
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Glutathione (GSH) is synthesised during oocyte maturation and represents the oocyte’s main non-enzymatic defence against oxidative stress. Inadequate defence against oxidative stress may be related to poor embryo quality and viability. In the present study, bovine oocytes were matured in vitro in the presence of GSH ethyl ester (GSH-OEt), a cell permeable GSH donor, and its effects on subsequent fertilisation and embryo development were assessed. GSH-OEt significantly increased the GSH content of IVM oocytes without affecting fertilisation or Day 3 cleavage rates. Maturation in the presence of GSH-OEt did not significantly increase the blastocyst rate compared with control oocytes. However, 5 mM GSH-OEt treatment resulted in significantly higher blastocyst total cell number. The GSH level of IVM oocytes was significantly decreased in the absence of cumulus cells and when cumulus–oocyte complexes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine oocytes and restored the rate of normal fertilisation, but not embryo development, to levels seen in control oocytes. Thus, GSH-OEt represents a novel approach for effective in vitro elevation of bovine oocyte GSH and improvement in blastocyst cell number.
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Uh, Kyungjun, Alayna Hay, Paula Chen, Emily Reese y Kiho Lee. "Design of novel oocyte activation methods: the role of zinc". Biology of Reproduction 106, n.º 2 (22 de diciembre de 2021): 264–73. http://dx.doi.org/10.1093/biolre/ioab235.
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Abstract Oocyte activation occurs at the time of fertilization and is a series of cellular events initiated by intracellular Ca2+ increases. Consequently, oocytes are alleviated from their arrested state in meiotic metaphase II (MII), allowing for the completion of meiosis. Oocyte activation is also an essential step for somatic cell nuclear transfer and an important tool to overcome clinical infertility. Traditional artificial activation methods aim to mimic the intracellular Ca2+ changes which occur during fertilization. Recent studies emphasize the importance of cytoplasmic Zn2+ on oocyte maturation and the completion of meiosis, thus suggesting artificial oocyte activation approaches that are centered around the concentration of available Zn2+in oocytes. Depletion of intracellular Zn2+ in oocytes with heavy metal chelators leads to successful oocyte activation in the absence of cellular Ca2+ changes, indicating that successful oocyte activation does not always depends on intracellular Ca2+ increases. Current findings lead to new approaches to artificially activate mammalian oocytes by reducing available Zn2+ contents, and the approaches improve the outcome of oocyte activation when combined with existing Ca2+-based oocyte activation methods. Here, we review the important role of Ca2+ and Zn2+ in mammalian oocyte activation and development of novel oocyte activation approaches based on Zn2+ availability.
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Pedersen, H. S., Y. Liu, R. Li, S. Purup, P. Løvendahl, P. Holm, P. Hyttel y H. Callesen. "Selection of pre- versus postpubertal pig oocytes for parthenogenetic activation and somatic cell nuclear transfer". Reproduction, Fertility and Development 27, n.º 3 (2015): 544. http://dx.doi.org/10.1071/rd13283.
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Pig oocytes have been used increasingly for in vitro production techniques in recent years. The slaughterhouse-derived oocytes that are often used are mostly of prepubertal origin. The aims of the present study were to compare the developmental competence between pre- and postpubertal pig oocytes, and to develop a simple and practical method for the selection of prepubertal pig oocytes for parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) based on oocyte morphology after IVM and oocyte inside zona pellucida (ZP) diameter (‘small’ ≤110 µm; ‘medium’ >110 µm; ‘large’ ≥120 µm). Meiotic competence and blastocyst rates after PA and SCNT of prepubertal oocytes increased with oocyte size, with the large prepubertal oocytes reaching a level similar to postpubertal oocytes after SCNT. Blastocyst cell number was not related to oocyte inside ZP diameter and oocyte donor to the same extent as blastocyst rate. Very low blastocyst rates were obtained after PA of morphologically bad pre- and postpubertal oocytes. In conclusion, measurement of inside ZP diameter combined with morphological selection is useful to remove incompetent oocytes. Further studies are needed to clarify the relative importance of cytoplasmic volume and stage in oocyte growth phase.
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Chen, Ying, Wendy N. Jefferson, Retha R. Newbold, Elizabeth Padilla-Banks y Melissa E. Pepling. "Estradiol, Progesterone, and Genistein Inhibit Oocyte Nest Breakdown and Primordial Follicle Assembly in the Neonatal Mouse Ovary in Vitro and in Vivo". Endocrinology 148, n.º 8 (1 de agosto de 2007): 3580–90. http://dx.doi.org/10.1210/en.2007-0088.
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In developing mouse ovaries, oocytes develop as clusters of cells called nests or germ cell cysts. Shortly after birth, oocyte nests dissociate and granulosa cells surround individual oocytes forming primordial follicles. At the same time, two thirds of the oocytes die by apoptosis, but the link between oocyte nest breakdown and oocyte death is unclear. Although mechanisms controlling breakdown of nests into individual oocytes and selection of oocytes for survival are currently unknown, steroid hormones may play a role. Treatment of neonatal mice with natural or synthetic estrogens results in abnormal multiple oocyte follicles in adult ovaries. Neonatal genistein treatment inhibits nest breakdown suggesting multiple oocyte follicles are nests that did not break down. Here we investigated the role of estrogen signaling in nest breakdown and oocyte survival. We characterized an ovary organ culture system that recapitulates nest breakdown, reduction in oocyte number, primordial follicle assembly, and follicle growth in vitro. We found that estradiol, progesterone, and genistein inhibit nest breakdown and primordial follicle assembly but have no effect on oocyte number both in organ culture and in vivo. Fetal ovaries, removed from their normal environment of high levels of pregnancy hormones, underwent premature nest breakdown and oocyte loss that was rescued by addition of estradiol or progesterone. Our results implicate hormone signaling in ovarian differentiation with decreased estrogen and progesterone at birth as the primary signal to initiate oocyte nest breakdown and follicle assembly. These findings also provide insight into the mechanism of multiple oocyte follicle formation.
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Yamazaki, Yukiko, Teruhiko Wakayama y Ryuzo Yanagimachi. "Contribution of cumulus cells and serum to the maturation of oocyte cytoplasm as revealed by intracytoplasmic sperm injection (ICSI)". Zygote 9, n.º 4 (noviembre de 2001): 277–82. http://dx.doi.org/10.1017/s0967199401001307.
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The fertilisability and developmental capacity of mouse oocytes matured in vitro were examined by in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI). While more than 50% of cumulus-enclosed oocytes were fertilised by IVF after maturation in serum-supplemented medium, none were fertilised when the oocytes matured without serum. By ICSI, the majority (78-94%) of the oocytes were fertilised regardless of the presence or absence of serum in oocyte maturation media. Although the majority (88-92%) of cumulus-free germinal vesicle oocytes underwent nuclear maturation in both serum-free and serum-containing media, those matured in the presence of serum were more readily fertilised by ICSI (43%) than those matured without it (3-5%). The cumulus-free oocytes co-cultured with cumulus cells but without serum were fertilised at 36%, suggesting some secreted factor promotes the oocyte's cytoplasmic maturation. The oocytes fertilised by ICSI developed into normal-term fetuses regardless of the presence or absence of serum or cumulus cells in oocyte maturation medium. These results lead us to conclude that (a) the cytoplasm of the oocytes can mature in serum-free medium and (b) the presence of both the serum and the cumulus cells in the medium surrounding maturing oocytes is beneficial for the development of the fertilisation- and development-competence of oocyte cytoplasm.
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Maside, Carolina, Irene Sánchez-Ajofrín, Daniela Medina-Chávez, Benner Alves, José Julián Garde y Ana Josefa Soler. "Oocyte Morphometric Assessment and Gene Expression Profiling of Oocytes and Cumulus Cells as Biomarkers of Oocyte Competence in Sheep". Animals 11, n.º 10 (27 de septiembre de 2021): 2818. http://dx.doi.org/10.3390/ani11102818.
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Oocyte quality is crucial for subsequent embryo development and so it is a major challenge in assisted reproductive technologies. The aim of the present work was to evaluate the morphometric parameters of oocytes (experiment 1) and the relative gene expression of oocytes and cumulus cells (CCs) (experiment 2) as biomarkers of oocyte quality after individually culturing them (one oocyte or embryo/drop). In experiment 1, individually matured oocytes were measured and classified into small, intermediate, and large oocytes after a cluster analysis, based on total diameter (with zona pellucida, ZP), oocyte diameter (without ZP), and ZP thickness. These oocytes were individually fertilized in vitro and cultured. The embryo development was evaluated up to the blastocyst stage. According to the total diameter, oocyte diameter, and ZP thickness, the blastocyst rate decreased in the small oocytes group (3.1 ± 3.1, 14.1 ± 9.4, and 26.7 ± 3.9, respectively) compared to the intermediate (29.4 ± 5.2, 30.5 ± 10.1, and 28.6 ± 9.6, respectively) and large oocytes groups (54.2 ± 13.5, 44.4 ± 3.9, and 67.6 ± 12.4, respectively). In addition, the probability of reaching the blastocyst stage was positively related to the total diameter (p < 0.001), oocyte diameter (p < 0.05), and ZP thickness (p < 0.001). Furthermore, the relative gene expression of BAX, BCL2, GDF9, and GJA1 was lower in oocytes classified as large. In experiment 2, the mRNA transcript relative abundance pattern of genes in CCs was evaluated according to oocyte total diameter and developmental stage reached. CCs from oocytes classified as large and oocytes capable of developing to the blastocyst stage had a lower relative expression of BAX, STAR, and PTGS2, while a higher expression of HAS2 and SDC2 transcript was observed for those oocytes. In conclusion, oocyte morphometric parameters and gene expression analysis in oocytes and CCs provide methods for the identification of the most competent oocytes for assisted reproductive technologies in sheep.
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Uozumi, T. y H. Funahashi. "272 INTRACELLULAR NITRIC OXIDE LEVEL OF PORCINE OOCYTES IS NEGATIVELY CORRELATED WITH OOCYTE MATURATION RATE AND CUMULUS EXPANSION INDEX IN A CHEMICALLY DEFINED MEDIUM". Reproduction, Fertility and Development 23, n.º 1 (2011): 234. http://dx.doi.org/10.1071/rdv23n1ab272.
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Nitric oxide (NO) has been known to inhibit nuclear maturation in cumulus–enclosed oocytes in rodents. The objective of this study was to examine if meiotic stimulators, such as dibutyryl cAMP and epidermal growth factor (EGF), influence intracellular NO level of oocytes and if the level is correlated with oocyte maturation rate and cumulus expansion in a chemically defined medium. Oocyte–cumulus complexes (OCC) were aspirated from mid-size follicles (3–6 mm in diameter) of prepuberal porcine ovaries. The OCC were cultured in modified porcine oocyte medium with various supplements – gonadotropins plus dibutyryl cAMP (Gn + cAMP), EGF plus dibutyryl cAMP (EGF + cAMP), dibutyryl cAMP alone (cAMP), EGF alone (EGF), and non-supplements (none) – for a first 20-h period and then in fresh porcine oocyte medium (without those supplements) for another 24 h in an atmosphere of 5% CO2 in air at 39°C. Following in vitro maturation culture, OCC were assessed for the degree of cumulus expansion (scored from 0 as cumulus free to 5 as full expansion) and then additionally cultured with DAF2-DA, an indicator of NO, for an additional 1-h period in the same condition. The oocytes were denuded with 0.1% hyaluronidase, and the intensity of fluorescence was measured. The oocytes were also fixed, stained with acetic orcein, and observed for meiotic stage. Statistical analysis was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). Maturation rates and cumulus expansion indexes were significantly affected by various supplement conditions (Table 1). The intensity of fluorescence showing intracellular NO level was also different among experimental groups (Table 1). A negative correlation was found between intracellular NO intensity and maturation rate (r2 = 0.71) or cumulus expansion index (r2 = 0.70). From these results, we conclude that there is a synergistic effect of cAMP and EGF on cumulus expansion and oocyte maturation and the reduction of oocyte NO levels in a chemically defined medium. Furthermore, a reduction of oocyte NO level seems to be included in the induction of cumulus expansion and oocyte maturation. Table 1.Effects of supplements on nuclear maturation, cumulus expansion, and intracellular NO level of porcine oocytes1
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Fernandes, C. A. F., P. G. V. Oliveira, C. H. B. Oliveira, F. H. V. Hazin y P. Travassos. "Oocyte development and fecundity type of the Brazilian Snapper Lutjanus alexandrei Moura & Lindeman, 2007 (Perciformes: Lutjanidae)". Brazilian Journal of Biology 76, n.º 1 (22 de enero de 2016): 126–35. http://dx.doi.org/10.1590/1519-6984.14714.
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Abstract Lutjanid species exhibit multiple spawning behaviour during an extended spawning season in warm months, asynchronous oocyte development and indeterminate fecundity. Although early studies have contributed to knowledge of the reproductive cycle of many species within the group, they have not considered aspects about the number of cortical alveoli oocyte stage throughout maturity phases along spawning season. The latter aspect is also considered very important to confirm indeterminate fecundity hypothesis. In the present study, were analyzed 154 Brazilian snapper Lutjanus alexandrei female gonads obtained from artisanal fisheries in Pernambuco State (Brazil) between October 2010 and March 2011. Were measured oocyte size frequency distribution for maturity phases (developing, spawning capable and actively spawning), and oocyte development stage (unyolked oocytes, cortical alveoli, primary, secondary and tertiary vitellogenic oocytes and hydrated oocytes), and also the oocyte stage frequency during spawning season. The frequency of cortical alveoli oocyte stage was constantly found in the spawning period (>37%), showing slight variation throughout maturity phases. The absence of gap in the oocyte size frequency distribution between primary and secondary oocyte growth stages during spawning season is a strong indicator of continuous oocyte recruitment from reserve stocks. In addition, co-occurrence of tertiary vitellogenic oocytes, hydrated oocytes, post-ovulatory follicles and yellow-brown bodies in the histological sections of ovaries reinforce indeterminate fecundity hypothesis.
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Chen, Jing, Maggie M. Chi, Kelle H. Moley y Stephen M. Downs. "cAMP pulsing of denuded mouse oocytes increases meiotic resumption via activation of AMP-activated protein kinase". REPRODUCTION 138, n.º 5 (noviembre de 2009): 759–70. http://dx.doi.org/10.1530/rep-08-0535.
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cAMP plays a critical role in the control of oocyte maturation, as a high level of cAMP maintains oocyte arrest at the first meiotic prophase. Yet this study shows that pulsing meiotically arrested denuded oocytes (DO) with cAMP induces oocyte maturation through the activation of AMP-activated protein kinase (PRKA). Short-term (3 h) pulsing of meiotically arrested oocytes with forskolin, an adenyl cyclase (AC) activator, increased oocyte cAMP, led to elevated AMP, and induced oocyte meiotic resumption compared to oocytes continuously cultured in the control medium with or without forskolin. Western analysis showed that germinal vesicle (GV)-stage oocytes after forskolin pulsing contained increased levels of phospho-acetyl CoA carboxylase (pACACA), a primary substrate of PRKA. Pulsing oocytes with the phosphodiesterase (PDE)-sensitive cAMP analog, 8-bromo-cAMP (8-Br-cAMP), also increased pACACA and pPRKA levels in GV-stage oocytes and induced oocyte meiotic resumption. Moreover, the PRKA inhibitors, compound C and araA, prevented 8-Br-cAMP pulsing-induced maturation. The lack of effect on meiotic induction and PRKA activation when oocytes were pulsed with the PDE-resistant activators of cAMP-dependent protein kinase, Sp-cAMP-AM and Sp-5,6-DCI-cBIMPS, suggests that cAMP degradation is required for pulsing-induced maturation. Pulsing oocytes with the exchange protein directly activated by cAMP (Epac)-specific activator, 8-CPT-2′-O-Me-cAMP, had no stimulatory effect on oocyte maturation, suggesting Epac is not involved in the pulsing-induced maturation. Taken together, these data support the idea that a transient increase in oocyte cAMP can induce meiotic resumption via activation of PRKA.
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Lisle, R. S., K. Anthony, M. A. Randall y F. J. Diaz. "Oocyte–cumulus cell interactions regulate free intracellular zinc in mouse oocytes". REPRODUCTION 145, n.º 4 (abril de 2013): 381–90. http://dx.doi.org/10.1530/rep-12-0338.
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Zinc increases in the oocyte during maturation and is required for progression and completion of meiosis. The objective of this study was to determine whether cumulus cells regulate the levels of free intracellular zinc in the oocyte during maturation. In the cumulus–oocyte complex (COC) the relative level of free intracellular zinc was almost fourfold higher in cumulus cells compared with the resident germinal vesicle-stage oocyte. Removal of cumulus cells caused a fourfold increase in intracellular zinc in the oocyte by 1 h after cumulus cell removal, but subsequent coculture of denuded oocytes with COC decreased free intracellular zinc in the oocyte by 65%. Thus, cumulus cells suppress free intracellular zinc in the oocyte. The mRNA transcripts for the zinc transporter proteins Slc39a6, Slc39a8, Slc39a9, Slc39a10, Slc39a12, Slc30a2, Slc30a4, Slc30a5 and Slc30a8 mRNAs were higher in oocytes, while Slc39a1, Slc39a7, Slc39a13, Slc39a14, Slc30a6, Slc30a7 and Slc30a9 mRNAs were higher in cumulus cells. Thus a complex zinc transport network is present in the COC. Pretreatment with epidermal growth factor for 4 h abolished the ability of COCs to restrict free intracellular zinc in denuded oocytes. Coculture of denuded metaphase II oocytes with COC lowers free intracellular zinc in mature oocytes. Oocytes matured in vivo or oocytes from older mice had lower levels of free intracellular zinc than oocytes matured in vitro or from younger mice. Thus, a precise mechanism for regulating oocyte zinc homeostasis has been uncovered in the COC that is disrupted with increasing age or by removal of cumulus cells.
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Wang, Qiang, Maggie M. Chi, Tim Schedl y Kelle H. Moley. "An intercellular pathway for glucose transport into mouse oocytes". American Journal of Physiology-Endocrinology and Metabolism 302, n.º 12 (15 de junio de 2012): E1511—E1518. http://dx.doi.org/10.1152/ajpendo.00016.2012.
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Glucose is an essential nutrient for mammalian cells. Emerging evidence suggests that glucose within the oocyte regulates meiotic maturation. However, it remains controversial as to whether, and if so how, glucose enters oocytes within cumulus-oocyte complexes (COCs). We used a fluorescent glucose derivative (6-NBDG) to trace glucose transport within live mouse COCs and employed inhibitors of glucose transporters (GLUTs) and gap junction proteins to examine their distinct roles in glucose uptake by cumulus cells and the oocyte. We showed that fluorescent glucose enters both cumulus-enclosed and denuded oocytes. Treating COCs with GLUT inhibitors leads to simultaneous decreases in glucose uptake in cumulus cells and the surrounded oocyte but no effect on denuded oocytes. Pharmacological blockade of of gap junctions between the oocyte and cumulus cells significantly inhibited fluorescent glucose transport to oocytes. Moreover, we find that both in vivo hyperglycemic environment and in vitro high-glucose culture increase free glucose levels in oocytes via gap junctional channels. These findings reveal an intercellular pathway for glucose transport into oocytes: glucose is taken up by cumulus cells via the GLUT system and then transferred into the oocyte through gap junctions. This intercellular pathway may partly mediate the effects of high-glucose condition on oocyte quality.
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Abe, H., H. Shiku, S. Aoyagi, T. Matsue y H. Hoshi. "319 OXYGEN CONSUMPTION OF BOVINE CUMULUS CELLS AND OOCYTES CULTURED IN DIFFERENT CULTURE SYSTEMS FOR OOCYTE MATURATION". Reproduction, Fertility and Development 18, n.º 2 (2006): 267. http://dx.doi.org/10.1071/rdv18n2ab319.
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Oxygen consumption is a ubiquitous parameter that can provide valuable information on metabolic mechanisms and on oocyte and embryo quality. Recently, we succeeded in non-invasively and quantitatively determining oxygen consumption of individual bovine embryos by scanning electrochemical microscopy (SECM). The aim of this study was to assess by SECM the oxygen consumption of bovine cumulus cells and oocytes cultured in serum-free and serum-supplemented media for oocyte maturation. Bovine cumulus–oocyte complexes (COCs) were obtained from ovarian follicles 2–6 mm in diameter. COCs were cultured in IVMD101 medium for serum-free culture and HPM199 medium supplemented with 5% calf serum (HPM199+CS) for serum-supplemented culture in a humidified atmosphere of 5% CO2 in air (20% O2) at 38.5°C for 24 h. Oxygen consumption by single COCs was non-invasively quantified by a SECM measuring system (Abe et al. 2004 J. Mamm. Ova Res. 21, 22). After the measurements, COCs were treated with 0.5% pronase to completely remove the cumulus cells. The oxygen consumption of single denuded oocyte was measured by SECM. Some COCs and oocytes were prepared for transmission electron microscopy. Oxygen consumption has been monitored in COCs and oocytes cultured in IVMD101 and HPM199+CS media for oocyte maturation (Table 1). Oxygen consumption rates of the immature COCs and denuded oocytes (immediately upon recovery from ovary: control) were 6.91 and 0.70 (×10−14 mol s−1), respectively. In serum-free culture (IVMD101), an increase in oxygen consumption rate was found in oocytes, whereas the oxygen consumption of COCs decreased during oocyte maturation. On the other hand, the oxygen consumption of COCs and oocytes cultured in serum-supplemented medium (HPM199+CS) were not change compared with that of controls. Electron microscopic study demonstrated that the mitochondria moved from a peripheral location in the ooplasm to an even spatial distribution in the oocytes cultured in IVMD101 medium, whereas many of the mitochondria in oocytes cultured in HPM199+CS were distributed in the peripheral region of the ooplasm after oocyte maturation. These results suggest that the respiration activity of bovine cumulus cells and oocytes changed during oocyte maturation, and the respiration activity and ultrastructural features of oocytes may affect the culture conditions. The SECM procedures may provide valuable information on oocyte quality and culture conditions for oocyte maturation. Table 1. Oxygen consumption rates (F × 10−14 mol s−1) of the bovine COCs and oocytes in oocyte maturation cultures
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Guo, Jing, Teng Zhang, Yueshuai Guo, Tao Sun, Hui Li, Xiaoyun Zhang, Hong Yin et al. "Oocyte stage-specific effects of MTOR determine granulosa cell fate and oocyte quality in mice". Proceedings of the National Academy of Sciences 115, n.º 23 (21 de mayo de 2018): E5326—E5333. http://dx.doi.org/10.1073/pnas.1800352115.
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MTOR (mechanistic target of rapamycin) is a widely recognized integrator of signals and pathways key for cellular metabolism, proliferation, and differentiation. Here we show that conditional knockout (cKO) of Mtor in either primordial or growing oocytes caused infertility but differentially affected oocyte quality, granulosa cell fate, and follicular development. cKO of Mtor in nongrowing primordial oocytes caused defective follicular development leading to progressive degeneration of oocytes and loss of granulosa cell identity coincident with the acquisition of immature Sertoli cell-like characteristics. Although Mtor was deleted at the primordial oocyte stage, DNA damage accumulated in oocytes during their later growth, and there was a marked alteration of the transcriptome in the few oocytes that achieved the fully grown stage. Although oocyte quality and fertility were also compromised when Mtor was deleted after oocytes had begun to grow, these occurred without overtly affecting folliculogenesis or the oocyte transcriptome. Nevertheless, there was a significant change in a cohort of proteins in mature oocytes. In particular, down-regulation of PRC1 (protein regulator of cytokinesis 1) impaired completion of the first meiotic division. Therefore, MTOR-dependent pathways in primordial or growing oocytes differentially affected downstream processes including follicular development, sex-specific identity of early granulosa cells, maintenance of oocyte genome integrity, oocyte gene expression, meiosis, and preimplantation developmental competence.
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Curnow, E. C., J. Ryan, D. Saunders y E. S. Hayes. "241 STRATEGIES TO IMPROVE GLUTATHIONE CONTENT OF IN VITRO-MATURED BOVINE OOCYTES". Reproduction, Fertility and Development 20, n.º 1 (2008): 200. http://dx.doi.org/10.1071/rdv20n1ab241.
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Glutathione is the main non-enzymatic defense against oxidative stress and a critical part of oocyte maturation and normal fertilization. Our aim was to test different strategies to manipulate cellular glutathione (GSH) content of bovine in-vitro-matured (IVM) oocytes and study the development of embryos produced from such oocytes. The reducing agents lipoic acid (LA, intracellular) and dihydrolipoic acid (DHLA, extracellular) were compared to the cell-permeable reduced glutathione (GSH) donor glutathione ethyl ester (OET) for their effect on oocyte GSH content, oocyte maturation, and blastocyst development (OET only). Reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. Cumulus–oocyte complexes (COCs) were aspirated from abattoir-derived ovaries and matured for 24 h in a humidified atmosphere of 6% CO2 at 38.5�C in modified tissue culture medium (mTCM199) supplemented with 2% (LA, DHLA) or 10% (OET) fetal calf serum (FCS; Gibco, Grand Island, NY, USA), 0.1 IU bLH and 0.1 IU bFSH (Sioux Biochemicals, Sioux City, IA, USA). COCs were matured in the presence of either LA (100 µm) or DHLA (100 µm) alone or in combination with L-cystine (CYS; 0.6 mm), CYS alone, or OET at 1, 3, and 5 mm. COCs matured under control and experimental conditions were denuded of cumulus cells (40 IU hyaluronidase) and scored for maturity. GSH content of MII oocytes was determined by colorimetric assay (Northwest Life Science Specialties, LLC, Vancouver, WA, USA). Oocytes matured in OET were inseminated with frozen/thawed bull sperm (2 � 106 mL-1), cultured to the blastocyst stage (COOK bovine medium, COOK Australia, Brisbane, Queensland, Australia), and subjected to differential cell count (propidium iodide/Hoechst). GSH levels (mean � SEM) and developmental data (percentage) are expressed for n = 18–73 oocytes or embryos and were analyzed by ANOVA or chi-square test (significance, P ≤ 0.05). LA alone failed to increase oocyte GSH content over 2% FCS control levels (6.98 � 0.22 pmol/oocyte v. 5.26 � 0.4 pmol/oocyte). DHLA alone significantly increased oocyte GSH content (9.64 � 0.8 pmol/oocyte) compared to both LA and controls (10% FCS; 4.78 � 0.36 pmol/oocyte). CYS alone (10.18 � 0.58 pmol/oocyte) or in combination with LA (10.84 � 0.37 pmol/oocyte) or DHLA (9.75 � 0.66 pmol/oocyte) significantly increased GSH compared to controls. GSH content of MII oocytes matured in 5 mm OET (8.35 � 0.35 pmol/oocyte) was significantly higher compared to control (5.07 � 0.32 pmol/oocyte), 1 mm (4.21 � 0.18 pmol/oocyte), and 3 mm (7.12 � 0.35 pmol/oocyte) OET treatments. Maturation rates of oocytes were significantly reduced in 2% FCS (51.1–72%) compared to 10% FCS (90.5%). OET treatment (1–5 mm) did not significantly alter maturation rate compared to control (75–89.8%). Blastocyst development of IVM oocytes treated with 1 mm OET (22.5%) was significantly lower compared to 3 mm (42.3%) and 5 mm (41.1%) OET but not to control (33.6%). Blastocysts from IVM oocytes treated with 5 mm OET had significantly higher cell counts compared to controls (126 � 6.4 cells v. 100.8 � 5.2 cells). Bovine IVM is a valuable model for testing the efficacy of various strategies to increase oocyte cellular GSH. Both strategies improve oocyte GSH levels, and an increase in blastocyst cell number occurred with GSH donor treatment (5 mm OET).
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Ali, Abbas Musa y Saad Akram Hatif. "Low oocyte quality related with the aging ewes". Iraqi Journal of Veterinary Medicine 37, n.º 2 (31 de diciembre de 2013): 261–65. http://dx.doi.org/10.30539/ijvm.v37i2.1391.
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This study was conducted to know the effect of ewe age on oocyte quality as well as the relationsbetween oocyte viability and normal uterine condition. Eighty three (83) reproductive systems ofnon-pregnant ewes were collected from Al-shulla abattoir. The Total oocytes were aspirated from right ovaries reached 61.45% and 38.55% from left ovaries. Immediately after aspiration, the oocytes were examined by light microscopic and conceded as mature if surrounded completely with cumulus oopherus. While the stained oocytes by trypan blue were conceded as dead oocytes and excluded. According to ewes age the oocytes were classified into (3) groups, the first group ranged between 1-2 years, second group 3-6 years and the third group over 6 years. The total oocyte collection fromthese groups was 20, 23, 40 oocyte. The results indicated that 14 oocytes (70%), 17(73.91%) and 10(25%) from groups 1, 2, and 3 with cumulus cells, respectively. While the total live oocyte reached to 60. Normal endometrium was observed in 90%, 95% and 80%for 1,2and 3 groups respectively. It was concluded from this study that aged ewes showed low quality oocyte with infertile endometrium.
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Lu, Yujie, Yue Zhang, Jia-Qian Liu, Peng Zou, Lu Jia, Yong-Teng Su, Yu-Rong Sun y Shao-Chen Sun. "Comparison of the toxic effects of different mycotoxins on porcine and mouse oocyte meiosis". PeerJ 6 (20 de junio de 2018): e5111. http://dx.doi.org/10.7717/peerj.5111.
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Background Aflatoxin B1 (AFB1), deoxynivalenol (DON), HT-2, ochratoxin A (OTA), zearalenone (ZEA) are the most common mycotoxins that are found in corn-based animal feed which have multiple toxic effects on animals and humans. Previous studies reported that these mycotoxins impaired mammalian oocyte quality. However, the effective concentrations of mycotoxins to animal oocytes were different. Methods In this study we aimed to compare the sensitivity of mouse and porcine oocytes to AFB1, DON, HT-2, OTA, and ZEA for mycotoxin research. We adopted the polar body extrusion rate of mouse and porcine oocyte as the standard for the effects of mycotoxins on oocyte maturation. Results and Discussion Our results showed that 10 μM AFB1 and 1 μM DON significantly affected porcine oocyte maturation compared with 50 μM AFB1 and 2 μM DON on mouse oocytes. However, 10 nM HT-2 significantly affected mouse oocyte maturation compared with 50 nM HT-2 on porcine oocytes. Moreover, 5 μM OTA and 10 μM ZEA significantly affected porcine oocyte maturation compared with 300 μM OTA and 50 μM ZEA on mouse oocytes. In summary, our results showed that porcine oocytes were more sensitive to AFB1, DON, OTA, and ZEA than mouse oocytes except HT-2 toxin.
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Jiang, Yao, Yingting He, Xiangchun Pan, Penghao Wang, Xiaolong Yuan y Bin Ma. "Advances in Oocyte Maturation In Vivo and In Vitro in Mammals". International Journal of Molecular Sciences 24, n.º 10 (21 de mayo de 2023): 9059. http://dx.doi.org/10.3390/ijms24109059.
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The quality and maturation of an oocyte not only play decisive roles in fertilization and embryo success, but also have long-term impacts on the later growth and development of the fetus. Female fertility declines with age, reflecting a decline in oocyte quantity. However, the meiosis of oocytes involves a complex and orderly regulatory process whose mechanisms have not yet been fully elucidated. This review therefore mainly focuses on the regulation mechanism of oocyte maturation, including folliculogenesis, oogenesis, and the interactions between granulosa cells and oocytes, plus in vitro technology and nuclear/cytoplasm maturation in oocytes. Additionally, we have reviewed advances made in the single-cell mRNA sequencing technology related to oocyte maturation in order to improve our understanding of the mechanism of oocyte maturation and to provide a theoretical basis for subsequent research into oocyte maturation.
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Li, Yong-Hai, Yi Hou, Wei Ma, Jin-Xiang Yuan, Dong Zhang, Qing-Yuan Sun y Wei-Hua Wang. "Localization of CD9 in pig oocytes and its effects on sperm–egg interaction". Reproduction 127, n.º 2 (febrero de 2004): 151–57. http://dx.doi.org/10.1530/rep.1.00006.
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CD9 is a cell surface protein that participates in many cellular processes, such as cell adhesion. Fertilization involves sperm and oocyte interactions including sperm binding to oocytes and sperm–oocyte fusion. Thus CD9 may play an essential role during fertilization in mammals. The present study was conducted to examine whether CD9 is present in porcine gametes and whether it participates in the regulation of sperm–oocyte interactions. The presence of CD9 in ovarian tissues, oocytes and spermatozoa was examined by immunohistochemistry, immunofluorescence and immunoblotting. Sperm binding and penetration of oocytes treated with CD9 antibody were examined by in vitro fertilization. The results showed that CD9 was present on the plasma membrane of oocytes at different developmental stages. A 24 kDa protein was found in oocytes during in vitro maturation by immunoblotting and its quantity was significantly (P < 0.001) increased as oocytes underwent maturation and reached the highest level after the oocytes had been cultured for 44 h. No positive CD9 staining was found in the spermatozoa. Both sperm binding to ooplasma and sperm penetration into oocytes were significantly (P < 0.01) reduced in anti-CD9 antibody-treated oocytes (1.2 ± 0.2 per oocyte and 16.6% respectively) as compared with oocytes in the controls (2.5 ± 0.4 per oocyte and 70.3% respectively). These results indicated that CD9 is expressed in pig oocytes during early growth and meiotic maturation and that it participates in sperm–oocyte interactions during fertilization.
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Hipp, H. S., A. J. Gaskins, Z. P. Nagy, S. M. Capelouto, D. B. Shapiro y J. B. Spencer. "Effect of oocyte donor stimulation on recipient outcomes: data from a US national donor oocyte bank". Human Reproduction 35, n.º 4 (6 de marzo de 2020): 847–58. http://dx.doi.org/10.1093/humrep/deaa003.
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Abstract STUDY QUESTION How does ovarian stimulation in an oocyte donor affect the IVF cycle and obstetric outcomes in recipients? SUMMARY ANSWER Higher donor oocyte yields may affect the proportion of usable embryos but do not affect live birth delivery rate or obstetric outcomes in oocyte recipients. WHAT IS KNOWN ALREADY In autologous oocyte fresh IVF cycles, the highest live birth delivery rates occur when ~15–25 oocytes are retrieved, with a decline thereafter, perhaps due to the hormone milieu, with super-physiologic estrogen levels. There are scant data in donor oocyte cycles, wherein the oocyte environment is separated from the uterine environment. STUDY DESIGN, SIZE, DURATION This was a retrospective cohort study from 2008 to 2015 of 350 oocyte donors who underwent a total of 553 ovarian stimulations and oocyte retrievals. The oocytes were vitrified and then distributed to 989 recipients who had 1745 embryo transfers. The primary outcome was live birth delivery rate, defined as the number of deliveries that resulted in at least one live birth per embryo transfer cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS The study included oocyte donors and recipients at a donor oocyte bank, in collaboration with an academic reproductive endocrinology division. Donors with polycystic ovary syndrome and recipients who used gestational carriers were excluded. The donors all underwent conventional ovarian stimulation using antagonist protocols. None of the embryos underwent pre-implantation genetic testing. The average (mean) number of embryos transferred to recipients was 1.4 (range 1–3). MAIN RESULTS AND THE ROLE OF CHANCE Per ovarian stimulation cycle, the median number of oocytes retrieved was 30 (range: 9–95). Among the 1745 embryo transfer cycles, 856 of the cycles resulted in a live birth (49.1%). There were no associations between donor oocyte yield and probability of live birth, adjusting for donor age, BMI, race/ethnicity and retrieval year. The results were similar when analyzing by mature oocytes. Although donors with more oocytes retrieved had a higher number of developed embryos overall, there was a relatively lower percentage of usable embryos per oocyte warmed following fertilization and culture. In our model for the average donor in the data set, holding all variables constant, for each additional five oocytes retrieved, there was a 4% (95% CI 1%, 7%) lower odds of fertilization and 5% (95% CI 2%, 7%) lower odds of having a usable embryo per oocyte warmed. There were no associations between donor oocyte yield and risk of preterm delivery (<37 weeks gestation) and low birthweight (<2500 g) among singleton infants. LIMITATIONS, REASONS FOR CAUTION Ovarian stimulation was exclusively performed in oocyte donors. This was a retrospective study design, and we were therefore unable to ensure proportional exposure groups. These findings may not generalizable to older or less healthy women who may be vitrifying oocytes for planned fertility delay. There remain significant risks to aggressive ovarian stimulation, including ovarian hyperstimulation. In addition, long-term health outcomes of extreme ovarian stimulation are lacking. Lastly, we did not collect progesterone levels and are unable to evaluate the impact of rising progesterone on outcomes. WIDER IMPLICATIONS OF THE FINDINGS Live birth delivery rates remain high with varying amounts of oocytes retrieved in this donor oocyte model. In a vitrified oocyte bank setting, where oocytes are typically sent as a limited number cohort, recipients are not affected by oocyte yields. STUDY FUNDING/COMPETING INTEREST(S) Additional REDCap grant support at Emory was provided through UL1 TR000424. Dr. Audrey Gaskins was supported in part by a career development award from the NIEHS (R00ES026648).
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Mohd Faizal, Ahmad, Yodo Sugishita, Yuki Suzuki-Takahashi, Hideyuki Iwahata, Seido Takae, Yuki Horage-Okutsu y Nao Suzuki. "Twenty-first century oocyte cryopreservation—in vitro maturation of immature oocytes from ovarian tissue cryopreservation in cancer patients: A systematic review". Women's Health 18 (enero de 2022): 174550572211142. http://dx.doi.org/10.1177/17455057221114269.
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Objectives: Our review aimed to consolidate the latest update on the application of in vitro maturation among immature oocyte harvest in combination with ovarian tissue cryopreservation known as ovarian tissue oocyte–in vitro maturation. Methods: A thorough search for relevant studies was conducted via PubMed, Google Scholar, EMBASE, and clinical.gov databases up to December 2020. The primary outcome was the oocyte maturation rate, which measured the number of immature oocytes (geminal vesicle stage) that progressed to mature oocytes (meiosis II stage) following in vitro maturation. The secondary outcomes were the fertilization rate following intracytoplasmic sperm injection/in vitro fertilization of these oocytes for the embryo cryopreservation cohort. Our review included pre-pubertal girls and women with cancer who underwent ovarian tissue oocyte–in vitro maturation as fertility preservation. Results: The primary search identified 207 studies. Twelve manuscripts were selected for inclusion in our review following duplication assessment, title and abstract screening, and full-text evaluation tailored to our inclusion criteria. All the population belonged to a cancer group and underwent concurrent ovarian tissue oocyte–in vitro maturation. A total of 5724 immature oocytes were obtained following ovarian tissue cryopreservation. Approximately 33.84% of the immature oocytes successfully matured via in vitro maturation, which were cryopreserved as oocytes or fertilized as embryos and subsequently stored for future use. Conclusion: Our review proposed the potential application of ovarian tissue oocyte–in vitro maturation in increasing the number of mature oocytes. The acceptable improvement in oocyte maturation rate following in vitro maturation indicates that improving oocyte outcomes is an excellent cost-effective strategy for fertility preservation among women with cancer.
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Finn, Roderick Nigel, Gunn C. Østby, Birgitta Norberg y Hans Jørgen Fyhn. "In vivo oocyte hydration in Atlantic halibut (Hippoglossus hippoglossus); proteolytic liberation of free amino acids, and ion transport, are driving forces for osmotic water influx". Journal of Experimental Biology 205, n.º 2 (15 de enero de 2002): 211–24. http://dx.doi.org/10.1242/jeb.205.2.211.
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SUMMARY The in vivo swelling and hydration of maturing oocytes of Atlantic halibut Hippoglossus hippoglossus were studied in order to characterise the osmotic mechanism underlying oocyte hydration in oviparous marine teleosts that spawn pelagic eggs. Sequential biopsies from two females, spanning four hydration cycles, were examined by osmometry, solute analysis and electrophoresis of dissected hydrating oocytes and ovulated eggs. The hydration cycle of the biopsied halibuts lasted 33–54 h. The majority of ovarian oocytes existed in a pre-hydrated condition (individual wet mass approx. 3.7 mg, diameter approx. 1.87 mm, 63 % H2O) with easily visible, non-coalesced, yolk platelets. Group-synchronous batches of the pre-hydrated oocytes increased in individual wet mass, diameter and water content to reach the ovulated egg stage of approximately 15 mg, 3.0 mm and 90 % H2O, respectively. The yolk osmolality of the hydrating oocytes was transiently hyperosmotic to the ovarian fluid (range 305–350 mOsmol l–1) with a peak osmolality of about 450 mOsmol l–1 in oocytes of 6–8 mg individual wet mass. The transient hyperosmolality was well accounted for by the increase in oocyte content of free amino acids (FAAs; approx. 2300 nmol oocyte–1), K+ (approx. 750 nmol oocyte–1), Cl– (approx. 900 nmol oocyte–1), total ammonium (approx. 300 nmol oocyte–1) and inorganic phosphate (Pi; approx. 200 nmol oocyte–1) when relating to the increase in cellular water. The oocyte content of Na+ did not increase during the hydration phase. Extensive proteolysis of yolk proteins, in particular a 110 kDa protein, correlated with the increase in the FAA pool, although the latter increased by approx. 20 % more than could be accounted for by the decrease in the oocyte protein content. Both indispensable and dispensable amino acids increased in the FAA pool, and particularly serine, alanine, leucine, lysine, glutamine and glutamate. Taurine content remained stable at approx. 70 nmol oocyte–1 during oocyte hydration. The results show that final hydration of Atlantic halibut oocytes is caused by an osmotic water uptake in which FAAs, derived mainly from the hydrolysis of a 110 kDa yolk protein, contribute approximately 50 % of the yolk osmolality and ions (Cl–, K+, Pi, NH4+) make up the balance.
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Rodriguez, Karina F. y Charlotte E. Farin. "Gene transcription and regulation of oocyte maturation". Reproduction, Fertility and Development 16, n.º 2 (2004): 55. http://dx.doi.org/10.1071/rd03078.
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The developmental potential of an embryo is dependent on the developmental potential of the oocyte from which it originates. The process of oocyte maturation is critical for the efficient application of biotechnologies such as in vitro embryo production and mammalian cloning. However, the overall efficiency of in vitro maturation remains low because oocytes matured in vitro have a lower developmental competence than oocytes matured in vivo. Furthermore, oocytes that have been exposed to gonadotropins have greater developmental competence than oocytes matured in the absence of gonadotropins. By understanding the molecular mechanisms underlying gonadotropin-induced maturation, improvement in oocyte maturation technologies may be expected as procedures to manipulate specific factors involved in signalling for resumption of meiosis are identified. The present review will focus on transcriptional mechanisms underlying the maturation of mammalian oocytes in vitro, as well as on the acquisition of oocyte developmental competence. In addition, a working model for the transcriptional control of mammalian oocyte maturation is proposed.
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Tanaka, M., J. D. Hennebold, J. Macfarlane y E. Y. Adashi. "A mammalian oocyte-specific linker histone gene H1oo: homology with the genes for the oocyte-specific cleavage stage histone (cs-H1) of sea urchin and the B4/H1M histone of the frog". Development 128, n.º 5 (1 de marzo de 2001): 655–64. http://dx.doi.org/10.1242/dev.128.5.655.
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Oocytes and early embryos of multiple (non-mammalian) species lack the somatic form of the linker histone H1. To the best of our knowledge, a mammalian oocyte-specific linker (H1) histone(s) has not, as yet, been reported. We have uncovered the cDNA in question in the course of a differential screening (suppression subtractive hybridization (SSH)) project. Elucidation of the full-length sequence of this novel 1.2 kb cDNA led to the identification of a 912 bp open reading frame. The latter encoded a novel 34 kDa linker histone protein comprised of 304 amino acids, tentatively named H1oo. Amino acid BLAST analysis revealed that H1oo displayed the highest sequence homology to the oocyte-specific B4 histone of the frog, the respective central globular (putative DNA binding) domains displaying 54% identity. Substantial homology to the cs-H1 protein of the sea urchin oocyte was also apparent. While most oocytic mRNAs corresponding to somatic linker histones are not polyadenylated (and remain untranslated), the mRNAs of (non-mammalian) oocyte-specific linker histones and of mammalian H1oo, are polyadenylated, a process driven by the consensus signal sequence, AAUAAA, detected in the 3′-untranslated region of the H1oo cDNA. Our data suggest that the mouse oocyte-specific linker histone H1oo (1) constitutes a novel mammalian homolog of the oocyte-specific linker histone B4 of the frog and of the cs-H1 linker histone of the sea urchin; (2) is expressed as early as the GV (PI) stage oocyte, persisting into the MII stage oocyte, the oocytic polar bodies, and the two-cell embryo, extinction becoming apparent at the four- to eight-cell embryonic stage; and (3) may play a key role in the control of gene expression during oogenesis and early embryogenesis, presumably through the perturbation of chromatin structure.
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Piekarski, Nadine, Theodore R. Hobbs, Darla Jacob, Tiah Schwartz, Fernanda C. Burch, Emily C. Mishler, Jared V. Jensen, Sacha A. Krieg y Carol B. Hanna. "A Comparison of Oocyte Yield between Ultrasound-Guided and Laparoscopic Oocyte Retrieval in Rhesus Macaques". Animals 13, n.º 19 (26 de septiembre de 2023): 3017. http://dx.doi.org/10.3390/ani13193017.
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Obtaining quality oocytes is a prerequisite for ART-based studies. Here we describe a method for transabdominal ultrasound-guided (US) oocyte retrieval in rhesus macaques (Macaca mullata) and compare it to the standard surgical approach using laparoscopy (LAP). We analyzed oocyte yield from six continuous reproductive seasons (2017–2023) that included n = 177 US-guided and n = 136 laparoscopic oocyte retrievals. While the ultrasound-guided technique retrieved significantly fewer oocytes on average (LAP: 40 ± 2 vs. US: 27 ± 1), there was no difference in the number of mature metaphase II oocytes (MII) between the two techniques (LAP: 17 ± 1 vs. US: 15 ± 1). We show that oocytes retrieved by the ultrasound-guided approach fertilize at the same rates as those obtained via the laparoscopic procedure (LAP Fert Rate: 84% ± 2% vs. US Fert Rate: 83% ± 2%). In conclusion, minimally invasive ultrasound-guided oocyte retrieval improves animal welfare while delivering equivalent numbers of mature oocytes, which are ideal for ART. Furthermore, we show that oocyte competency, as represented by fertilization rate, is not affected by retrieval technique. Therefore, the Oregon National Primate Research Center (ONPRC) has adopted the ultrasound-guided approach as the standard technique for oocyte retrieval.
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Herrick, Jason R. "Reversible meiotic arrest in feline oocytes". Reproduction, Fertility and Development 26, n.º 2 (2014): 258. http://dx.doi.org/10.1071/rd12341.
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Increasing intracellular concentrations of cyclic adenosine monophosphate (cAMP) within the cumulus–oocyte complex (COC) inhibits or delays spontaneous oocyte maturation and improves the developmental competence of the oocyte in many species, but information for carnivores is limited. The objectives of the present study were to describe the effects of isobutyl methylxanthine (IBMX), which decreases cAMP degradation, and forskolin, which increases cAMP production, on spontaneous and induced maturation (by equine chorionic gonadotrophin (eCG) and epidermal growth factor (EGF)) of feline oocytes and to evaluate the reversibility of IBMX-induced arrest by measuring the resumption of meiosis and embryonic development following IVF. IBMX decreased (P < 0.05) the incidence of spontaneous (6.7% vs 42.0%, metaphase II (MII)) and induced (5.6% vs 66.1% MII) maturation after 24 h of culture. In contrast, forskolin stimulated meiosis (81.7% MII; P < 0.05). Following 12 h of culture with IBMX and an additional 24 h with eCG and EGF in the absence of IBMX, the proportions of oocytes reaching MII (66.1%), cleaving (79.9%) and developing to the blastocyst stage (15.3%) were similar (P > 0.05) to oocytes cultured continuously with eCG and EGF (70.2%, 83.0% and 18.1%, respectively). These results demonstrate that IBMX reversibly inhibits both spontaneous and eCG+EGF-induced meiosis in feline oocytes without compromising the oocyte’s developmental competence.
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Astbury, Pia, Goutham N. Subramanian, Jessica Greaney, Chris Roling, Jacqui Irving y Hayden A. Homer. "The Presence of Immature GV− Stage Oocytes during IVF/ICSI Is a Marker of Poor Oocyte Quality: A Pilot Study". Medical Sciences 8, n.º 1 (16 de enero de 2020): 4. http://dx.doi.org/10.3390/medsci8010004.
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Here we investigate whether the presence of germinal vesicle-stage oocytes (GV− oocytes) reflects poor oocyte developmental competence (or quality). This was a prospective, non-randomised, cohort pilot-study involving 60 patients undergoing in vitro fertilization/ intracytoplasmic sperm injection for whom complete pregnancy outcome data were available. Patients in whom GV− oocytes were retrieved (GV+) at transvaginal oocyte retrieval (TVOR) were compared with those from whom no GVs were retrieved (GV−). We found that GV+ (n = 29) and GV− (n = 31) patients were similarly aged (35.4 vs. 36.4 years; p = 0.446). GV+ patients had a mean of 2.41 ± 2.03 GVs and comparable yields of MII oocytes to GV− patients (11 ± 6.88 vs. 8.26 ± 4.84; p = 0.077). Compared with GV− patients, GV+ patients had markedly lower implantation rates (11.8% vs. 30.2%; p = 0.022) as well as oocyte utilisation rates for clinical pregnancy (2.3% vs. 6.8%; p = 0.018) and live-birth (1.9% vs. 5.7%; p = 0.029). DNA damage levels measured using γH2AX immunostaining were not different in oocytes from women <36 years versus those ≥36 years (p = 0.606). Thus, patients who have GV− stage oocytes at TVOR exhibit poor oocyte quality reflected in reduced per-oocyte pregnancy success rates and uniformly high levels of oocyte DNA damage.
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Petersen, Morten R., Michael Hansen, Birthe Avery y Ingrid B. Bøgh. "A Method for Chronological Intravital Imaging of Bovine Oocytes during In Vitro Maturation". Microscopy and Microanalysis 14, n.º 6 (6 de noviembre de 2008): 549–60. http://dx.doi.org/10.1017/s1431927608080872.
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AbstractOocyte maturation is known to affect the chances for successful fertilization, embryonic development, establishment of pregnancy and delivery of a live, healthy, and viable offspring. Two-photon laser scanning microscopy (TPLSM) has previously been used to evaluate early embryonic development without a detectable impairment of subsequent development, but has never been applied to assess mammalian oocytes throughout in vitro maturation (IVM). Visualization of structures within live oocytes during IVM, followed by fertilization and embryo culture, may improve the understanding of oocyte maturation. To visualize structures within bovine oocytes using TPLSM, it is necessary to remove the cumulus cells that normally surround the oocyte during maturation. Repeated visualization of structures within the same oocyte is possible, if movement of the oocyte can be avoided. In this article, we describe the development of a method for repeated intravital imaging of denuded bovine oocytes using an upright TPLSM equipped with a specially constructed incubator. Oocytes were stained with Hoechst 33258, and the nuclear structures were evaluated. Oocyte fertilization rate was not affected by TPLSM exposure, but the developmental capacity of the denuded oocytes was significantly reduced. This is, to our knowledge, the first article describing repeated intravital imaging during mammalian oocyte maturation using TPLSM.
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Andreu-Vázquez, C., F. López-Gatius, I. García-Ispierto, M. J. Maya-Soriano, R. H. F. Hunter y M. López-Béjar. "Does heat stress provoke the loss of a continuous layer of cortical granules beneath the plasma membrane during oocyte maturation?" Zygote 18, n.º 4 (24 de marzo de 2010): 293–99. http://dx.doi.org/10.1017/s0967199410000043.
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SummaryThe objective of the present study was to evaluate the influence of heat stress on bovine oocyte maturation. Both nuclear stage and distribution of cortical granules (CG) were simultaneously evaluated in each oocyte. Oocyte overmaturation under standard conditions of culture was also evaluated. For this purpose, logistic regression procedures were used to evaluate possible effects of factors such as heat stress, overmaturation, replicate, CG distribution and metaphase II (MII) morphology on oocyte maturation. Based on the odds ratio, oocytes on heat stressed (HSO) and overmaturated (OMO) oocyte group were, respectively, 14.5 and 5.4 times more likely to show anomalous MII morphology than those matured under control conditions (CO). The likelihood for an oocyte of showing the CG distribution pattern IV (aging oocyte) was 6.3 and 9.3 times higher for HSO and OMO groups, respectively, than for the CO group. The risk of undergoing anomalous oocyte maturation, considering both nuclear stage and distribution of CG was 17.1 and 18 times greater in oocytes cultured in HSO and OMO groups, respectively, than those in the CO group. In conclusion, heat stress proved to be valuable in aging oocytes. Heat stress advanced age for nuclear and cytoplasmic processes in a similar form to that of oocyte overmaturation.
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Oliveira, Reginaldo Luis, Katia Cristina Silva-Santos, Suellen Miguez Gonzalez, Camila Bizarro-Silva, Fernanda Zandonadi Machado, Ana Paula Frederico Rodrigues Loureiro Bracarense y Marcelo Marcondes Seneda. "Proliferative activity of oocytes in multi-oocyte follicles of bovine ovary". Semina: Ciências Agrárias 38, n.º 6 (23 de noviembre de 2017): 3591. http://dx.doi.org/10.5433/1679-0359.2017v38n6p3591.
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We characterized the proliferative activity of multi-oocyte follicles with anti-nuclear antigen of proliferating cells (PCNA). Ovaries (n = 12) from heifers were processed for histology. From 789 multi-oocytes follicles observed, only 11 were considered appropriated for immunostaining, since they presented all nuclei of the oocytes clearly visible. All multi-oocyte follicles were positive for PCNA, but some oocytes showed no proliferative activity. We conclude that oocytes in multi-oocyte follicles seem to be in different stages of the cell cycle.
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Servou, Eleni, Eudoxia Schismenou y Stylianos Somarakis. "Quantitative Analysis of Ovarian Dynamics of European Sardine Sardina pilchardus (Walbaum, 1792) during Its Spawning Period". Fishes 8, n.º 5 (25 de abril de 2023): 226. http://dx.doi.org/10.3390/fishes8050226.
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Fish with indeterminate fecundity spawn multiple times throughout a protracted reproductive period. During that period several ovulation events succeed one another, and different oocyte developmental stages co-occur in the ovaries with new oocytes consistently recruiting from one growth phase to the next to form the sequential batches. In this study, we examined in detail the oocyte recruitment and development pattern of the sequential batches in a commercially important fish with indeterminate fecundity, the European sardine. The numbers and sizes of oocytes at different developmental stages were estimated for four phases of the ovulatory cycle (ovarian stages) and during the main spawning season (November–March) by applying the oocyte packing density theory in combination with stereological techniques. General linear models (GLMs) were used to test for changes in oocyte sizes as well as relative oocyte numbers per developmental stage within the different ovarian stages in the successive spawning months. A temporal association between several transition events of the oocyte development process was revealed. Specifically, the final maturation of the advanced batch triggered (a) the recruitment of oocytes from primary to secondary growth phase, (b) de novo vitellogenesis and (c) a surge of yolk deposition in primary vitellogenic oocytes. Oocyte recruitment was completed two days after the ovulation of the advanced batch and relative numbers of primary and secondary growth oocytes were thereafter stable until the next final maturation event. This pattern of oocyte recruitment and growth remained unchanged during the course of the spawning season. This study advances our knowledge on oocyte recruitment and development in fish with indeterminate fecundity, which is key to understanding reproduction and its drivers at the individual and population level.
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Cai, L., E. Kim, S. U. Hwang, J. D. Yoon, Y. Jeon, E. Lee y S. H. Hyun. "156 EFFECTS OF MORPHOLOGY TYPE OF POLAR BODY ON PORCINE OOCYTE QUALITY AND DEVELOPMENTAL POTENTIAL AFTER IN VITRO FERTILIZATION". Reproduction, Fertility and Development 26, n.º 1 (2014): 192. http://dx.doi.org/10.1071/rdv26n1ab156.
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Evaluation of morphology of first polar body (1st PB) could be a method for the oocyte's quality and developmental competence. The developmental potential of oocyte with fragmented PB after in vitro maturation (IVM) is a controversial issue. The aim of this study is to investigate the effects of PB morphology type on oocyte quality and developmental competence after IVF. Porcine ovaries were obtained from prepubertal gilts at a local slaughterhouse and transported to the laboratory within 2 h in physiological saline supplemented with 100 IU mL–1 penicillin G and 100 mg mL–1 streptomycin sulfate. The cumulus–oocyte complexes (COC) were aspirated using an 18-gauge needle attached to a 10-mL disposable syringe from superficial follicles 3 to 6 mm in diameter followed by IVM. After IVM, oocytes were classified into 3 types as follows, oocytes with normal PB (A type), oocytes with a little of fragmented PB (B type), and oocytes with separated 2 PBs (C type), respectively. As classification of PB types, we analysed the distribution ratio of each PB type after IVM, and then performed IVF for analysis of fertilization rate and developmental potential. The ratio of oocyte with A type (73%) was significantly (P < 0.05) higher than that of B type (24.5%) or C type (2.5%) after IVM. Only mature oocytes were selected from A and B type and were subjected to IVF because of a small number of oocytes with C type. In the IVF experiment, the efficiency of monospermy and fertilization were significantly higher in oocytes of A type (46.7%) than those of type B (20.0%). The cleavage rate of oocytes with A type (63.9%) was significantly (P < 0.05) higher than the oocytes with B type (43.8%). Embryonic developmental competence to the blastocyst stage after IVF was significantly (P < 0.05) higher in the A-type oocytes (26.3%) than in the B-type oocytes (16.9%). The levels of glutathione and reactive oxygen species were not affected by the morphological classification of the PB. In summary, these results suggest that polar body morphology could be a marker of oocyte quality after IVM. We are currently studying gene expression of each oocytes and blastocysts. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.
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Абакушина, Е. В., Ю. В. Гельм y А. С. Миценык. "Флуоресцентный микроскопический анализ жизнеспособности ооцитов млекопитающих после витрификации-=SUP=-*-=/SUP=-". Журнал технической физики 126, n.º 5 (2019): 611. http://dx.doi.org/10.21883/os.2019.05.47660.9-19.
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AbstractThe paper reports the results of a fluorescent microscopy analysis of the viability of oocytes from cattle and pigs after vitrification. Oocytes were frozen in a vitrification media containing varying concentrations of cryoprotectors in several steps with subsequent vitrification. After cryobank storage for 14 days, experimental samples were thawed and oocyte viability was analyzed by oocyte morphology assessment and fluorescent microscopy. Two different kits were used to stain oocytes, one specific for necrosis/apoptosis (Propidium iodide/Alexa Fluor 488 Annexin) and the other specific for live/dead cells (Calcein-AM/ethidium homodimer-1). Fluorescent microscopy of porcine and bovine oocytes has demonstrated that the fluorescent dye Calcein-AM should be chosen to assess oocyte viability, since Propidium iodide and ethidium homodimer-1 do not represent the oocyte cell death. Therefore, Propidium iodide and ethidium homodimer-1 cannot serve as indicators of real oocyte death.
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MILLS, Catherine, Elizabeth SUTTON, Julian KOPLIN, Ezra KNEEBONE, Karinne LUDLOW y Ainsley NEWSON. "Mitochondrial Donation and Egg Donor Consent in Australia". Fertility & Reproduction 04, n.º 03n04 (septiembre de 2022): 150. http://dx.doi.org/10.1142/s2661318222740620.
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Background: Legislation to permit mitochondrial donation (MD) in Australia was introduced into Federal Parliament in early 2021, and the techniques may be legalized and made available soon. MD enables women affected by disease-causing mutations in their mitochondrial DNA to have a genetically related child who is unlikely to inherit these mutations. MD relies on the donation of oocytes. Australia’s oocyte donation system does not meet current demand for oocytes and MD would add to this. Consequently, the implementation of MD would raise critical questions about the system of procuring donors and using their oocytes. The proposed model for implementing MD in Australia does not address these issues. We address two key inter-related concerns – oocyte availability and donor consent – in how best to meet current and future demand for donor oocytes. Aim: To consider ethical, social, and regulatory issues arising from the proposed implementation of MD in Australia, particularly oocyte availability and donor consent issues. Method: We discuss the current system of oocyte donation in Australia and consider likely impacts of MD on this. We review alternative systems that have been proposed to enhance the availability of oocytes, focusing on ethical aspects of these with reference to specific features of the Australian context, donor consent, and MD. Results: MD will increase demand for oocytes if introduced in Australia. Alternative procurement systems to address the shortage of oocytes may be required. Refining the system of consent used for oocyte donation may be an important feature of increasing oocyte availability for MD. Conclusion: As Australia presses ahead with the potential implementation of MD, consideration should be given as to whether the current system for oocyte donation is adequate. We conclude that it is necessary to consider alternative systems for enhancing oocyte availability in Australia, which, in some circumstances, may include changing consent procedures.
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Yang, M., S. Hu, L. Cox, M. Regouski, H. Rutigliano, C. Isom y I. Polejaeva. "307 MATURATION RATE AND GENE EXPRESSION ANALYSIS OF GOAT OOCYTES SELECTED BY FOLLICLE SIZE AND BRILLIANT CRESYL BLUE STAINING". Reproduction, Fertility and Development 27, n.º 1 (2015): 242. http://dx.doi.org/10.1071/rdv27n1ab307.
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Oocyte quality plays a critical role in determining the success of embryo development. Studies on cattle and goats indicate that oocytes derived from large follicles (LFO) have greater developmental competence than those derived from small follicles (SFO). Brilliant cresyl blue (BCB) staining determines the activity of glucose-6-phosphate dehydrogenase and is a commonly used noninvasive marker of oocyte competence. Studies in pigs, goats, cows, mice, and dogs showed that the maturation and blastocyst developmental rate of BCB+ oocytes is significantly higher than BCB– oocytes. The aim of this study was to evaluate the maturation rate of goat oocytes selected based on follicular size and BCB staining and compare their relative patterns of gene expression. Maturation rate and gene expression profile were expected to be different in these oocyte groups. Cumulus-oocyte complexes were recovered from abattoir-derived ovaries using a slicing technique. Eleven rounds of oocyte maturation and 4 rounds of BCB staining were carried out. During each replicate, oocytes from large (≥3 mm) and small (<3 mm) follicles were collected separately from the same group of ovaries. Oocyte maturation rates were 54.3 ± 5.4% for LFO (n = 378) and only 33.5 ± 3.7% for SFO (n = 981; P < 0.01). The BCB+ (n = 223) oocytes yielded a significantly higher maturation rate than the BCB– (n = 194) oocytes (56.1 ± 1.8 v. 20.6 ± 3.8%, respectively; P < 0.001). Gene expression analysis was conducted on individual MII oocytes (21 oocytes per group). Specific target amplification was performed on a single oocyte directly by using the CellsDirect One-Step qRT–PCR Kit (Invitrogen). Quantitative real-time PCR was then performed using the 48.48 BioMark platform from Fluidigm. Forty two genes were selected from the following categories: growth factors, transcription factors, metabolism, pluripotency, cell cycle, apoptosis, and oocyte-specific genes. Relative expression values were calculated using the ΔΔCT (fold change) method and analysed by ANOVA. The significance was assigned at P < 0.05. The relative expression of CCNA2, CDK2, CCNB1, POU5F1, SOX2, EGF, FGF2, GDF9, ZP3, BCL2, GJA1, DDR1, PFKFB3, IGF2R, and GRB10 was significantly greater (P < 0.05) in both LFO and BCB+ oocytes compared to SFO and BCB– oocytes, respectively. The proapoptotic gene BAX, the ACSL3 gene involved in fatty acid oxidation, and the growth factor IGF1 were expressed significantly higher (P < 0.05) in SFO compared to LFO. By investigating these differentially expressed transcripts, we will better understand pathways involved in oocyte developmental competence and potentially use them as markers of oocyte quality. We expect that the ability to select oocytes of better quality based on BCB staining will improve outcomes of IVF and SCNT.
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Dumesic, Daniel A., Annie A. Guedikian, Vanessa K. Madrigal, Julia D. Phan, David L. Hill, Juan P. Alvarez y Gregorio D. Chazenbalk. "Cumulus Cell Mitochondrial Resistance to Stress In Vitro Predicts Oocyte Development During Assisted Reproduction". Journal of Clinical Endocrinology & Metabolism 101, n.º 5 (1 de mayo de 2016): 2235–45. http://dx.doi.org/10.1210/jc.2016-1464.
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Abstract Context: Complex cumulus cell-oocyte interactions govern energy utilization during oocyte development. Objective: This study investigates the relationship of cumulus cell mitochondria with oocyte development during ovarian stimulation for in vitro fertilization (IVF). Design: This is a prospective cohort study. Setting: The setting was an academic center. Patients: Thirty women underwent ovarian stimulation for IVF. Intervention(s): Pooled cumulus cells were collected; numbers of total and mature oocytes and two-pronuclear (day 1), six- to eight-cell cleavage (day 3), and blastocyst (day 5) embryos were recorded. Main Outcome Measure(s): A mitochondrial bioassay was developed with Jurkat cells and used with cumulus cells from IVF patients to correlate mitochondrial membrane potential resistance to carbonyl cyanide 3-chlorophenylhydrazone (CCCP) stress with oocyte development and embryogenesis. Results: Adjusting for FSH administered and maternal age, cumulus cell mitochondrial membrane potential resistance to CCCP positively correlated with numbers of total (P < .025) and mature (P < .025) oocytes retrieved. The highest oocyte numbers that correlated with cumulus cell mitochondrial membrane potential occurred in women with the greatest ovarian response to FSH (mitochondrial membrane potential resistance to CCCP-log FSH interactions: total oocytes P < .025; mature oocytes P < .05). Multiple regression modeling of mature oocyte numbers, age, and cumulus cell mitochondrial membrane potential resistance to CCCP showed that numbers of mature oocytes best correlated with numbers of embryos at all stages (P < .0001). Conclusion: During ovarian stimulation for IVF, cumulus cell mitochondrial membrane potential resistance to stress correlates with numbers of total and mature oocytes retrieved, suggesting that cumulus cell–oocyte interactions involving energy facilitate oocyte development.
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Kordowitzki, Paweł, Gabriela Sokołowska, Marta Wasielak-Politowska, Agnieszka Skowronska y Mariusz T. Skowronski. "Pannexins and Connexins: Their Relevance for Oocyte Developmental Competence". International Journal of Molecular Sciences 22, n.º 11 (31 de mayo de 2021): 5918. http://dx.doi.org/10.3390/ijms22115918.
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The oocyte is the major determinant of embryo developmental competence in all mammalian species. Although fundamental advances have been generated in the field of reproductive medicine and assisted reproductive technologies in the past three decades, researchers and clinicians are still trying to elucidate molecular factors and pathways, which could be pivotal for the oocyte’s developmental competence. The cell-to-cell and cell-to-matrix communications are crucial not only for oocytes but also for multicellular organisms in general. This latter mentioned communication is among others possibly due to the Connexin and Pannexin families of large-pore forming channels. Pannexins belong to a protein group of ATP-release channels, therefore of high importance for the oocyte due to its requirements of high energy supply. An increasing body of studies on Pannexins provided evidence that these channels not only play a role during physiological processes of an oocyte but also during pathological circumstances which could lead to the development of diseases or infertility. Connexins are proteins that form membrane channels and gap-junctions, and more precisely, these proteins enable the exchange of some ions and molecules, and therefore they do play a fundamental role in the communication between the oocyte and accompanying cells. Herein, the role of Pannexins and Connexins for the processes of oogenesis, folliculogenesis, oocyte maturation and fertilization will be discussed and, at the end of this review, Pannexin and Connexin related pathologies and their impact on the developmental competence of oocytes will be provided.
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Lee, Joohyeong, Eunhye Kim, Seon-Ung Hwang, Lian Cai, Mirae Kim, Hyerin Choi, Dongjin Oh, Eunsong Lee y Sang-Hwan Hyun. "Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs". Animals 11, n.º 4 (6 de abril de 2021): 1034. http://dx.doi.org/10.3390/ani11041034.
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This study aimed to examine the effects of treatment with glucuronic acid (GA) and N-acetyl-D-glucosamine (AG), which are components of hyaluronic acid (HA), during porcine oocyte in vitro maturation (IVM). We measured the diameter of the oocyte, the thickness of the perivitelline space (PVS), the reactive oxygen species (ROS) level, and the expression of cumulus cell expansion and ROS-related genes and examined the cortical granule (CG) reaction of oocytes. The addition of 0.05 mM GA and 0.05 mM AG during the first 22 h of oocyte IVM significantly increased oocyte diameter and PVS size compared with the control (non-treatment). The addition of GA and AG reduced the intra-oocyte ROS content and improved the CG of the oocyte. GA and AG treatment increased the expression of CD44 and CX43 in cumulus cells and PRDX1 and TXN2 in oocytes. In both the chemically defined and the complex medium (Medium-199 + porcine follicular fluid), oocytes derived from the GA and AG treatments presented significantly higher blastocyst rates than the control after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In conclusion, the addition of GA and AG during IVM in pig oocytes has beneficial effects on oocyte IVM and early embryonic development after PA and SCNT.
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Javed, Murid, Navid Esfandiari, Jason Swain y Essam Michael. "Human Oocyte Cryopreservation - An Emerging ART Technique: Are We Heading in the Right Direction?" Journal of Reproductive and Stem Cell Biotechnology 2, n.º 2 (diciembre de 2011): 109–20. http://dx.doi.org/10.1177/205891581100200205.
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Oocyte cryopreservation is a promising adjunct to human assisted reproductive technology. Slow rate freezing has been the cryopreservation standard for storage of sperm, embryos and, subsequently, oocytes. However, earlier concerns regarding damage to the meiotic spindle, loss of cortical granules and the low success rates as compared to the relative success of embryo cryopreservation caused little interest until the 1990s. Interest increased when many studies indicated that acceptable oocyte survival, in vitro fertilization, normal embryos and adequate blastocyst development can be achieved with oocyte cryopreservation. Recently introduced oocyte vitrification techniques are proving to be more efficient. Survival rates are close to 100% and developmental rates are similar to those achieved with fresh oocytes. This efficiency opens the way to the widespread application of the technique in various medical, legal, and social situations, even to replace embryo cryopreservation with the oocyte cryopreservation. Oocyte vitrification has dominated slow freezing to such an extent that it may soon become the exclusive cryopreservation choice, especially considering that potential disease transmission problems commonly associated with vitrification due to direct exposure of oocytes to liquid nitrogen can be eliminated by using the proper techniques and devices. Furthermore, cryopreservation of immature oocytes, ovarian follicles, ovarian tissue and whole ovary are other emerging technologies. Oocyte cryopreservation has tremendous opportunity for preserving fertility in cancer patients, for those who may not have sperm following oocyte retrieval and for those women who wish to delay their motherhood. The purpose of this article is to review the history of oocyte cryopreservation, its applications, current cryopreservation techniques and future trends for fertility cryopreservation, to determine if oocyte cryopreservation is proceeding in the right direction.
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Wang, Lingyan, Dexue Li y Ziyi Li. "Changes in the reciprocal position of the first polar body and oocyte chromosome set in golden hamsters". Bioscience Reports 29, n.º 5 (25 de junio de 2009): 315–20. http://dx.doi.org/10.1042/bsr20080104.
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The golden hamster is an attractive model organism for studying reproductive physiology, oncology, genetics and virology. In an effort to establish experimental protocols necessary for cloning golden hamsters, we examined changes in the reciprocal position of the FPB (first polar body) and chromosome set of MII (the second meiotic metaphase) oocytes of golden hamsters. Oocytes were collected under three different conditions: (i) oocyte direct recovery from the oviduct of hormonally treated donor; (ii) oocyte recovery from the oviduct of hormonally treated donor followed by 5 h/10 h in vitro culture; and (iii) oocyte recovery from ovaries of hormonally treated donors and in vitro maturation. Then oocyte recovery was performed from the oviduct of hormonally treated donors, followed by 5 h in vitro culture with colchicine and/or CB (cytochalasin B). Denuded oocytes were stained with Hoechst 33342 and propidium iodide and evaluated under a microscope. Our results demonstrate that the change in FPB position in relation to the MII oocyte chromosome set increases with age of in vivo-matured oocytes. Cumulus cells can protect the FPB of in vitro-cultured oocytes from degeneration but do not significantly affect its repositioning, and in vitro-matured oocytes age slower. The colchicine has a stronger effect on cytoplasmic protrusions of golden hamster oocytes when compared with CB. These results define conditions for changes in FPB position relative to the MII oocyte chromosome set. Early ovulated oocytes, in vitro-matured oocytes and oocytes treated with colchicine should improve the effectiveness of the cloning procedure in golden hamsters as an animal model for human diseases.
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Taketsuru, Hiroaki, Yuji Hirao, Naoki Takenouchi, Kosuke Iga y Takashi Miyano. "Effect of androstenedione on the growth and meiotic competence of bovine oocytes from early antral follicles". Zygote 20, n.º 4 (9 de noviembre de 2011): 407–15. http://dx.doi.org/10.1017/s0967199411000268.
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SummaryMedium that contains 17β-estradiol has been reported to support in vitro growth of bovine oocytes, isolated from early antral follicles, until the final stage. The aim of this study was to determine the effects of androstenedione in medium on such growing bovine oocytes. Oocyte–granulosa cell complexes were collected from early antral follicles and cultured for 14 days in medium supplemented with 17β-estradiol (0, 10 and 100 ng/ml) or androstenedione (0, 10 and 100 ng/ml). The mean diameter of oocytes measured after seeding on the culture substrate was 96.9 μm (n = 191). Either steroid was necessary for maintainance of the organization of oocyte–granulosa cell complexes over the 14-day culture period. In the 17β-estradiol- or the androstenedione-supplemented medium about 80% or 65%, respectively, of viable oocytes were recovered. In both groups the increase in oocyte size was significant after 14 days. The in vitro grown oocytes were cultured for a further 22–24 h for oocyte maturation; 13% and 30% of oocytes grown in the 10 and 100 ng/ml 17β-estradiol-supplemented medium reached metaphase II, respectively; more than 64% of oocytes grown in the androstenedione-supplemented medium matured to metaphase II. These results show that androstenedione, as 17β-estradiol, can maintain the viability of bovine oocyte–granulosa cell complexes and support the growth of oocytes, and that androstenedione promotes the acquisition of oocyte meiotic competence efficiently at a low dose.